emerging optical microscopy techniques for electrochemistry

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1 Emerging Optical Microscopy Techniques for Electrochemistry Jean-François Lemineur, a Hui Wang, b Wei Wang, b,* Frédéric Kanoufi a,* a Université de Paris, ITODYS, CNRS, 75006 Paris, France. b State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. * corresponding authors, e-mails: [email protected], [email protected]

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Page 1: Emerging Optical Microscopy Techniques for Electrochemistry

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Emerging Optical Microscopy Techniques for Electrochemistry

Jean-François Lemineur,a Hui Wang,b Wei Wang,b,* Frédéric Kanoufia,*

a Université de Paris, ITODYS, CNRS, 75006 Paris, France.

b State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210023, China.

* corresponding authors, e-mails: [email protected], [email protected]

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ABSTRACT

An optical microscope is probably the most intuitive, simple and commonly used

instrument to observe objects and discuss behaviors through images. Although the

idea of imaging electrochemical processes operando by optical microscopy was

initiated 40 years ago, it was not until significant progress made in the last two

decades in advanced optical microscopy or plasmonics that it could become a

mainstream electroanalytical strategy. This review illustrates the potential of different

optical microscopies to visualize and quantify local electrochemical processes with

unprecedented temporal and spatial resolution (below the diffraction limit), up to the

single object level with subnanoparticle or single molecule sensitivity. Developed

through optically and electrochemically active model systems, optical microscopy is

now shifting to materials and configurations focused on real-world electrochemical

applications.

KEYWORDS

Optical microscopy, electroanalysis, electrochemical conversion, single entity

electrochemistry, nanoparticles, operando imaging, spatiotemporal resolution

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1. INTRODUCTION

Electrochemistry is a vivid branch of science, particularly within the search for

renewable energy sources and systems enabling the conversion and storage of energy.

While great efforts have been made toward the synthesis and processing of

electroactive and electrocatalytic materials, often emphasizing the importance of their

structuring at the nanoscale, the improvement of the performance of most

electrochemical devices is hampered by the kinetic limitations of electrochemical

reactions. The understanding of their mechanisms and fundamentals relies on the

establishment of structure-function relationships, particularly at the nanoscale. This

has then driven the shift of traditional electroanalytical strategies and techniques

based on ensemble-averaged methods, e.g., current-potential, response toward the

imaging of electrochemical processes with higher sensitivity, spatial and temporal

resolution and manyfold complementary information.

Despite considerable progress in advanced in situ/operando characterization

techniques, optical microscopy remains the only technique that requires simple

operating procedures while being noninvasive and enabling multiple instrumental

couplings. Optical imaging of electrochemical processes was introduced in the

mid-1980s (1) along with scanning electrochemical microscopy. It was not until the

improvement of optical detectors (and components) and the development of

plasmonics that electroanalytical strategies employing optical microscopy were

brought back to the fore. The recent concepts of superlocalization, allowing to track

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phenomena with a resolution of a few nanometers which is lower than the smallest

picture element, i.e., the pixel, open many perspectives for imaging electrochemical

processes beyond the diffraction limit. Several reviews have detailed the general

operating principles and applications of such advanced optical microscopies in

(nano)chemistry, sometimes in electrochemistry (2–5). Herein, we summarize their

recent achievements in the imaging of multifarious electrochemical systems. After a

short description of some of the microscopes used, we show how they are currently

employed to resolve and quantify the heterogeneity of electrochemical interfaces,

from the macroscopic scale to the single nanoobject or even to the subentity or single

molecule level.

2. OPTICAL MICROSCOPES

A detailed description of the operating principle and configurations of the various

optical microscopes employed in electrochemistry can be found in (2–5). This review

mostly focuses on the use of wide-field microscopes, in which the light emanating

from a whole substrate is collected by a microscope objective and captured in a single

snapshot by a camera or a spectrograph for spectroscopic imaging. They offer higher

spatiotemporal resolution imaging than point scanning, e.g., confocal or tip-enhanced

Raman scattering, microscopes: within a single >50x50µm2 image snapshot, within

millisecond timescale, thousands of localized electrochemical behaviors can be

simultaneously obtained.

These microscopes are sensitive to the optical properties of the sample of interest,

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mostly absorbance, refractive index, scattering or luminescence. The growing

popularity of optical microscopy approaches in electrochemistry is not only related to

the ability to image but also the collection of quantitative information from the

mathematical treatment of the optical signal; see, e.g., (3). Plasmonic metals, e.g., Au

or Ag, constitute a highly sensitive detection tool in optics, as their interaction with

light induces the surface-confined oscillation of their free electrons known as surface

plasmon resonance (SPR). The SPR is strongly sensitive to the metal local charge

density or the refractive index of its environment, enabling different plasmonic-based

imaging of electrochemical processes. In SPR microscopy, the light locally reflected

by the interface between a thin layer of Au (used as an electrode) and an electrolyte

produces an SPR image sensitive to a wide variety of (electro)chemical reactions (2,

6). Localized SPRs, or LSPRs, are supported by plasmonic nanoparticles, NPs, or

nanostructures. Tracking the scattering (or LSPR) spectra of single plasmonic NPs

allows sensing single NP electrochemistry (2, 4, 7). The illumination of plasmonic

NPs or nanostructures (roughened electrodes) also produces a strong electromagnetic

near field able to enhance the Raman scattering generated by (individual) molecules

by several orders of magnitude (8, 9). The local increase in Raman intensity is used to

provide molecular vibrational images in surface-enhanced Raman scattering and

SERS microscopy with single molecule sensitivity.

Bright field and reflectivity microscopes mostly use (axial) illumination along the

objective axis and collect light transmitted or reflected by the sample of interest. They

can probe absorbance, refractive index or light emission, such as fluorescence or

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Raman scattering, after appropriate filtering of the excitation light beam. Oblique

incidence illuminations are used mostly for (i) dark-field configurations and (ii)

extreme total internal reflection (TIR) conditions, as they offer a lower optical

background level, enabling single nanoparticle or single molecule imaging sensitivity.

Dark-field illumination avoids blurring the detector, which only collects the light

scattered by the sample of interest. It has mostly been used to image the scattering of

plasmonic NPs with the eventual spectroscopic capture of their LSPR spectrum.

However, a broader class of scattering NPs has more recently been imaged at higher

sensitivity by interferometric scattering microscopes (10).

The TIR condition allows confined illumination (by evanescent waves) to light up

only objects located within a few hundred nm above the illuminated interface, which

is particularly useful for single-molecule fluorescence detection. Similar TIR

illumination conditions are used in plasmonic-based (SPR) microscopy.

Imaging without optical illumination, and therefore at the lowest optical background,

is possible using the electrochemical triggering of a luminescent reaction, named

electrochemiluminescence microscopy (5, 11, 12). As it involves chemically unstable

precursors of the luminescence reaction, electrochemiluminescence offers chemically

confined illumination of objects near the electrode and single photon sensitivity (13).

Finally, a microscope is characterized by two crucial notions: its sensitivity, i.e., its

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ability to detect an object, and its resolution, i.e., its ability to distinguish two objects

close together. Microscopes are diffraction limited, meaning that objects should be

separated by a distance greater than λ/2NA, with λ being the illumination wavelength

and NA being the objective numerical aperture. Furthermore, single objects smaller

than this limit, e.g., single NPs or single molecules, appear in an optical image as an

identical optical pattern, regardless of their structure or composition, named the point

spread function (PSF) or Airy disk. Note that the resolution of localization of optical

microscopes can be greatly improved by image posttreatment consisting of

approximating the PSF by a two-dimensional Gaussian distribution and

algorithmically extracting the spatial origin of a single emitter, also named its optical

center of mass or centroid. By superlocalization approaches, the location of various

electrochemical reactions is visualized operando at single nanoentities with a

resolution <5 nm.

3. OBJECTS OF STUDY

3.1 IMAGING OF HETEROGENEOUS INTERFACES

Probing nanoscale electrochemical events at heterogeneous interfaces discloses the

internal mechanism and detailed dynamics of electron transfer processes in analytical

chemistry and biosensing. Different imaging strategies have been developed to

visualize local electrochemical information at electrode interfaces, such as plasmonic,

electrochemiluminescence, and fluorescence microscopy (1–8). These revolutionary

studies reveal the intrinsic characteristics and mechanisms of nonfaradaic and redox

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processes with ultrasensitive temporal and spatial resolution. This section details the

optical electrochemical imaging of various heterogeneous interfaces.

3.1.1 Individual cells

Monitoring single-cell responses to substrates or small molecules and cellular

processes at the microscopic level deepens the understanding of the mechanisms of

physiological and biochemical dynamics. Optical techniques have been introduced to

study multifarious single-cell electron transfer events with high spatial resolution,

providing detailed information on their transient activities and local distributions (19,

20). Tao et al. first developed plasmonic-based electrochemical impedance

microscopy to uncover heterogeneous processes such as single-cell apoptosis and

electroporation with millisecond time resolution (21). A local impedance

measurement (𝑍) is derived from the local change in plasmonic intensity (∆𝜃) of a

thin Au SPR surface according to 𝑍!!(𝑥,𝑦,𝜔) = 𝑗𝜔𝛼∆𝜃(𝑥,𝑦,𝜔)/∆𝑉, where 𝜔 is

the angular frequency of the AC modulation, x and y are the locations on the electrode,

and 𝛼 is a constant determined by theoretical calculation or experimental calibration.

They optically resolved the local impedance of subcellular structures and ion

distributions in mammalian cells with submicrometer spatial resolution and excellent

detection sensitivity (~2 pS). By combining the electrochemical plasmonic impedance

imaging method with the traditional patch clamp technique (Figure 1a), the fast

propagation of the action potential in individual mammalian neurons was mapped

without any labeling (22). They further investigated the heterogeneous distribution of

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ion channels at the subcellular level and proposed studying various cellular

electrochemical activities and understanding the related biological functions and

mechanisms.

INSERT FIGURE 1

Recently, the groups of Sojic and Paolucci developed a surface-confined microscope

based on electrochemiluminescence illumination of objects and illustrated it to map

membrane adhesion sites of single cells on an electrode (20, 23). Their groups further

demonstrated the influence of photobleaching on electrochemiluminescence emission.

As both photo- and electrochemical activation involve the same excited state, the

more photoactivated the fluorophore is, the less active it is in the

electrochemiluminescence. Despite this issue, new imaging strategies combining

fluorescence recovery and electrochemiluminescence were envisioned (24).

3.1.2 Fingerprints

Visualizing latent fingerprints (LFPs) is an essential method for biometric identity

authentication. Various chemical and physical strategies have been explored to reveal

LFPs, including multimetal immunodeposition, fluorescence, and ink staining (25–30).

The ability of electrochemical techniques to identify explosive residues and other

chemicals secreted by LFPs has gradually gained attention. Tao et al. demonstrated a

plasmonic imaging technique combined with electrochemical measurement to map

human LFPs on an electrode surface (28). Finger secretions block the electron transfer

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process on the electrode, and the plasmonic contrast of the local fingerprint region is

transposed into a local electrochemical current of redox-active molecules in solution.

Later, Su and coauthors (Figure 1b) developed an electrochemiluminescence-based

imaging technique to enhance and visualize local LFPs using different dyes:

Ru(bpy)32+, rubrene, and electropolymerized luminol (25, 27, 29). The

electrochemiluminescence signal was generated only between the ridges of the LFPs,

and different details of the LFPs were resolved: the bifurcation, core, island, pore,

lake, peak, and termini.

Recently, Hu et al. reported a new strategy for transferring and imaging LFPs onto

nonporous substrates using simultaneous interfacial separation of a polydopamine

film and electroless silver deposition (27). As sweat components and underlying

substrates were well preserved, they generalized the approach to different substrates,

regardless of surface hydrophobicity or micromorphology.

3.1.3 Bipolar electrochemistry

A bipolar electrode (BPE) is a conductive material exposed to an external electric

field from the application of a potential difference between two electrodes in an

electrolyte. The potential difference induces electrical polarization at opposite poles

of the bipolar electrode, which manifests as a gradient in the distribution of free

electron density (31). When the potential difference is large enough, opposite

electrochemical reactions occur simultaneously at both ends of the BPE. As these

reactions occur without external current flow, their demonstration requires

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complementary visualizations, such as probe-labeled imaging with

electrochemiluminescence reagents, pH chromogens, or fluorescent dyes, and

label-free plasmonic imaging techniques (32–34). Crooks and coauthors developed

electrochemiluminescence-based imaging of BPE reactions. They provide a means to

locally quantify the thermodynamics and kinetics of the reactions to spatially

reconstruct the voltammogram of these reactions from an electrochemiluminescence

image. A triangular BPE is used, which allows, for electroanalysis, the quantification

of the reaction of interest on the smallest part of the BPE (toward the point of the

triangle) compared to the larger counterelectrode reaction part (35).

Xu and Chen reported an ultrasensitive wireless electrochemiluminescence biosensor

for quantitative monitoring of c-Myc target mRNA in tumor cells on a BPE substrate

(36). In this system-on-chip, they integrated RuSi@Ru(bpy)32+ for optical signal

amplification with a 24-fold improvement over Ru(bpy)32+-NHS labels. Beyond

electrochemiluminescence, Kuhn et al. successively presented other indirect imageries

of BPEs based on pH-triggered local precipitation (37) or fluorescence modulation

(32). The products of these reactions are monitored in the vicinity of the BPE.

Except for the above techniques using optical probes, plasmonic-based microscopy

provides label-free visualization and thus there is no need to engage a faradaic

reaction at the BPE a priori. Wang and coauthors first demonstrated the capability of

plasmonic imaging to directly visualize the interfacial potential distribution on a

bipolar microelectrode array with a sensitivity of 10 mV (33). The external electric

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field controls the redistribution of the free electron density on the BPE and thus

modifies its local dielectric (optical) properties. The local plasmonic response is thus

predicted using the Drude model. Furthermore, it is possible to locate the

zero-potential line on BPEs, where no reaction occurs, regardless of their geometry

(e.g., round, triangular, hexagonal, star, and diamond shapes) during nonfaradaic

charging and faradaic processes (38). The results revealed that the geometry of the

electrode and the nature and redox potential of the faradaic reactions affect the

position of the zero-potential line on the BPE.

3.1.4 Two-dimensional nanomaterials

Two-dimensional (2D) nanomaterials are emerging as novel platforms for

optoelectronics and biosensing due to their unique physical, chemical, and electronic

characteristics (39–41). The spatial charge distribution of these thin layers has been

studied by optical techniques, such as plasmonic, bright field, or interference

scattering microscopy, coupled with electrochemical measurements (6, 41–43). The

optical readout reveals fundamental electrochemical parameters of 2D electrodes and

their heterogeneity, such as quantum capacitance and local charge density, with high

spatial and temporal resolution. Graphene is the most studied 2D material without a

bandgap. Tao and coauthors mapped local electron and hole puddles with charged

impurities in a graphene monolayer by plasmon-based impedance microscopy (42).

The surface charge density, ∆𝜎, is related to the local interfacial capacity per unit

area ( 𝑐 ) and the applied potential (∆𝑉 ), according to ∆𝜎 = 𝑐 ∙ ∆𝑉 . Periodic

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modulations of the potential control the surface charge, resulting in a modulation of

the plasmonic intensity (∆𝜃 ). From the latter ∆𝜃 , extracted from the Fourier

transform of the graphene region images, a local capacitance distribution is obtained

according to 𝑐~∆𝜃/∆𝑉. Further charging induces an expansion of the graphene

according to Pauli repulsion. This expansion is imaged using a nm-sensitive optical

edge-tracking method (44). The technique allows determining the electromechanical

stress that increases quadratically with the modulation of the applied potential and

extracting the Young's modulus of different regions. Further oxidation of graphene at

potentials > 1.4 V results in its conversion to graphene oxide. This process was

imaged in situ by a label-free refractive index-sensitive optical technique such as

interference reflection microscopy (IRM). This reveals the formation of flower-like

patterns from which the local degree of graphene oxidation can be quantified and its

chemical vs. electrochemical oxidation compared (43).

Apart from graphene, molybdenum disulfide (MoS2) monolayers are another

attractive 2D material for next-generation nanoelectronic devices, with a direct

bandgap of 1.9 eV. Tao et al. imaged the local charge distribution of atomically thin

MoS2 upon electrochemical charging. The change in charge induces a local change in

the absorption of MoS2, which is imaged by bright-field transmission microscopy

with higher sensitivity (45).

3.1.5 Electrocorrosion and electrodeposition at a large interface

Electrocorrosion and electrodeposition are classical strategies for fabricating

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functional interfaces and improving the surface characteristics of metallic, ceramic, or

polymeric materials (46, 47). The basic principle of electrocorrosion and

electrodeposition is the destruction and formation of materials on a working electrode

immersed in an electrolyte solution and subjected to an external potential. The

morphological evolution of the electrode surface during these interfacial engineering

processes is essential to uncover the detailed dynamics and accurately determine

structure-function relationships. Different optical imaging techniques have been used

to probe corrosion processes, e.g., fluorescence, reflectivity or confocal microscopy.

They allow the identification of locally different reaction products or solution pH or

the identification and measurement of the size of crevices. V. Pérez-Herranz

developed a wide field reflectivity microscope allowing real-time observation at the

scale of several cm2 on copper and stainless steel electrode surfaces (48). They also

mapped the different corrosion behaviors of crevices and grain boundaries and

identified the generation of gas bubbles. Smyrl et al. used fluorescence microscopy to

image the regions where oxides, which trap fluorophores, preferentially form at

higher resolution. The measurement is complemented by confocal measurements

allowing a topographic (3D) image of crevices (49, 50). Vivier and coauthors

proposed a quantitative assessment by reflectivity imaging of the thickness of passive

layers during corrosion of carbon steel under polarization (51). They performed local

reflectivity measurements of the steel surface during cathodic and anodic

polarizations to study the formation of Fe2O3 and Fe3O4 oxides. It allows the

identification of the most active regions of the surface and the establishment,

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simultaneously over each µm2 regions of the mm2 imaged surface, of local

voltammograms of their activity or transformation.

These same imaging techniques are also used to follow operando the

electrodeposition processes at micrometric scales, in particular in energy storage or

conversion systems (52), to identify the formation of dendrites (53, 54) localized

operando at nanometer resolution (Figure 1c) or electrode passivation (55) and to

remedy them.

3.2 SINGLE NANO-ENTITY STUDIES

The growing use of nanoscale objects is bound to the identification of their intrinsic

properties, for which quantitative nanostructure-activity relationships are urgently

needed. In electrochemistry, different cross-reading approaches have been proposed at

the single NP level (56–59), mainly based on their isolation in time, one

electrochemical event at a time, or in space, by local electrochemical probing. In

addition to probing local electrochemistry with nanoelectrodes or nanopipettes, in the

so-called scanning electrochemical (SECM), electrochemical cell (SECCM) or ion

conductance (SICM) microscopies configurations, one can use optical microscopies

that allow high-sensitivity imaging and detection at the resolution of a single NP.

Recent developments proposed to integrate optical microscopy readout with such

scanning electrochemical microscopes (60, 61). The coupling of optical microscopy

with SECCM is of particular interest to image the subtle electrochemical processes

occurring inside the nano- or microelectrochemical cell, obtained by the confinement

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of a droplet of electrolyte by a nano- or micropipette. Indeed, in addition to high

resolution electrochemical imaging, SECCM allows a high throughput exploration of

local electrochemical processes by a versatile modifications or benchmarking in each

droplet of experimental parameters, e.g. surface or solution composition or

electrochemical interrogation. While SECCM enables nanoscale electrochemical

exploration with single droplet resolution, optical microscopy affords a

complementary subdroplet imaging resolution.

Coupling electrochemistry to optical microscopies appears relevant to probe operando

nanoscale electroactivities. Optical movies allow high-throughput readout of

individual NPs within large ensembles, altogether submitted to the same experimental

condition, allowing identification of subpopulation behaviors, for example by drawing

and comparing their individual electrochemical activity (e.g. current-potential,

charge-time, etc. curves). Moreover, NPs can be differentiated by their size, structure

or composition from their different optical properties, typically their optical cross

section (related to their refractive index, absorption, luminescence, etc.).

In the field of NP electrochemistry, optical microscopy has been applied to reveal and

study a wide variety of chemical or physical processes illustrated in Figure 2a for the

particular case of Ag-based NPs, i.e., the most studied system due to their plasmonic

activity and easily tunable (electro)chemical activity.

INSERT FIGURE 2

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3.2.1 Electrodeposition and electrodissolution

Optical monitoring of an electrode surface during electrodeposition reactions allows

simultaneous capture of the moment and location of formation of individual NPs with

a very large field of view (up to millions of NPs simultaneously (62)) and camera

temporal resolution (up to >1 kHz). Optical imaging then provides statistically

significant data to test and enrich NP nucleation/growth mechanisms and models.

For noble metals, e.g., Ag, the differences in the onset of NP appearance on the

electrode reflect the variability of their nucleation barrier (63, 64). Iron group metals

also reveal competition with other electrode reactions, such as water reduction in the

case of Ni or Co (65, 66). Beyond the local chemical information, localization of the

nucleation sites (67, 68) allowed reconstruction of each diffusion zone around the NPs

and probing diffusion cross-talk between neighboring sites.

Within a region of interest (ROI) centered on each NP, the transient evolution of the

local optical intensity is extracted from optical movies. Such transient gives insights

into the modes and kinetics of single-NP growth (69). This can be converted into the

amount of locally electrodeposited material (63, 64, 70) due to a calibration between

NP size and optical intensity obtained from the optical images of gauge NPs, ex situ

correlative SEM analysis or optical modeling. Combined with Faraday's law, local

currents, in the form of optovoltammograms (Figure 2b), associated with the

growth/dissolution of each NP are obtained by this quantitative analysis, again

evaluated for hundreds of NPs simultaneously.

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The reverse electrodissolution reaction was also studied for electrodeposited NPs (63,

64, 71) or nanocolloids immobilized on an electrode (69, 72, 73). The disappearance

of the optical feature associated with the NP in the images accounts for its dissolution

dynamics, investigated for metallic Ag NPs in different electrolytes (63, 64, 71).

Electrodeposition/stripping is an attractive strategy to decorate electrodes with a high

density of NPs of controlled size distribution, allowing the examination of

structure-activity relationships. The effect of NP size on their oxidation potentials,

observed for Ag NPs, validates Plieth's theory that links the electrochemical stability

of NPs (below ~50 nm) to their surface tension (63, 64).

The electrodissolution of Brownian nanocolloids was also probed by optical

microscopy. It allows tracking the motion of Brownian NPs in solution during their

collision (reactive or not) with a polarized electrode and complements electrochemical

nanoimpact experiments (56), in which the current spikes associated with a reactive

collision of NPs provide information on their size, dispersion, stability, concentration,

etc. The correlated optical and electrochemical detections revealed a more complex

picture. 3D optical tracking of Ag NPs near a polarized interface revealed intermittent

NP-electrode interactions associated with partial oxidation events (72, 74). This

supports the hypothesis of multistep Ag→Ag+ electrodissolution, first established

from high-frequency current traces and attributed to stochastic electrical

disconnection (75). Under conditions favoring Ag+ precipitation, a time lag is

observed between the injection of electrochemical charges and the dissolution of

optically probed NPs, highlighting solid phase conversion (e.g., to oxide, halide, or

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thiocyanate crystals (72, 73, 76–78)).

This type of optical imaging of the appearance or disappearance of optical features,

primarily used with model metal NPs, was extended to visualize and quantify in situ

the formation or dissolution of a variety of other materials, such as gas nanobubbles

(79–83) or ionic crystals (84), and holds promise for high-throughput monitoring of

structural deformations of nanoelectrocatalysts under operating conditions (85). The

technique can be easily extended to the study of various phase formation processes as

long as they can be triggered electrochemically, either by direct electrodeposition or

indirectly by local electrolysis.

3.2.2 Electrochemical conversion

During the electrochemical or redox conversion of an NP, the change in the redox

state is often associated with a change in its optical properties, such as fluorescence,

absorption, or scattering cross-section. Different optical microscopies can distinguish

the initial and final states of redox conversion of individual NPs. Gradual changes in

composition can even be monitored in situ and in real time, revealing mechanistic

pathways at the single NP level (Ag examples in Figure 2a).

The conversion of Ag-based metallic NPs into Ag+ salt nanocrystals is evidenced

under dark-field microscopy by a decrease in the intensity of the light they scatter

without, however, reaching total signal extinction. A spectrometer inserted at the end

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of the optical path, or a hyperspectral camera, completed the pure optical imaging

with their UV-vis spectrum, providing information on the composition and conversion

mechanisms in solutions of Ag+ precipitating agents (77, 78, 86–88).

The transformation of metallic NPs, e.g., Ag, into more noble metal NPs, e.g., Au, by

galvanic replacement, a popular redox reaction in colloidal synthesis, was followed in

situ under Au3+ solution flow by dark-field microscopy. The optical transients are also

characterized by a sudden drop in the scattering signal but observed after a waiting

time of variable duration. The broad distribution of waiting times confirms the

gradual transformation of solutions. The difference between single vs. ensemble NP

behaviors suggests that the transformation is kinetically limited by the stochastic

formation of a void in the NP lattice (broad distribution). Once the void is formed, the

NP transformation is rapid (sudden drop) and diffusion-limited (89). Other

mechanistic indications were identified, such as the role of precipitating Cl- or the NP

ligand shell (90, 91).

The methodology is applicable to nanomaterials used for energy storage or

conversion. The refractive index of LiCoO2 NPs decreases linearly with the amount of

Li-expelled ions, allowing imaging of their electrochemical (de)lithiation by refractive

index-sensitive microscopies (92). From the optical intensity fluctuations of

individual NPs recorded during cyclic lithiation/delithiation voltammetry or

nanoimpact experiments (93), Wang and coauthors quantified the dynamics of Li-ion

diffusion with an optically inferred current sensitivity of 50 fA.

Similarly, for supercapacitor applications, the insertion/deinsertion of alkali ions into

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electrochromic NPs, such as WO3 or Prussian blue (PB), was imaged under

bright-field transmission (94–98). The evolution of the light transmitted by individual

NPs (Figure 2c) analyzed according to the Beer-Lambert law allows quantification of

their conversion rate. For some WO3 NPs, slower and less complete insertion

dynamics, even more pronounced for NP aggregates, were observed, which suggests

irreversible trapping of Li+ at the NP-NP or NP-electrode interfaces. The

heterogeneity of ionic nanocrystal-electrode contacts has also been highlighted when

potassium ions are inserted into electrochromic PB nanocubes (99). Sputtering an

ultrathin layer of Pt onto electrode materials, as is often done in SEM, reconnects and

renders all nanocubes electroactive and avoids erroneous conclusions in establishing

structure-activity relationships.

3.2.3 Electrocatalytic systems

3.2.3.1. Probing molecular intermediates

Quantification of the electrocatalytic activity of single NPs is achieved by probing the

molecular products or intermediates of the reaction by fluorescence microscopy or

surface-excited Raman spectroscopy (SERS), sometimes with single-molecule

sensitivity (see Section 2.4). These microscopies mainly use a redox molecular probe

(commonly phenoxazine dyes such as resorufin), in which one of the redox forms is

luminescent or Raman active, or use pH-sensitive probes.

By adapting the strategy developed for photocatalysis (100), fluorescence microscopy

allowed imaging and evaluating (i) the deactivation of Pt/C electrocatalysts during the

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hydrogen oxidation reaction (101) or (ii) the 2- vs. 4-electron reduction pathways of

O2 by magnetite NPs (102).

Willets et al. imaged by SERS the local activity and distribution of reaction potentials

on Ag NP aggregates for the 2-electron conversion of Nile blue (103, 104). A recent

work suggests possible extension to the direct detection of valuable reaction products

such as CO2 and its reduction products (105).

Electrochemiluminescence involves redox and electrochemical reactions that can be

activated by Au NPs (106), resulting in NP visualization. Under the oxidizing

conditions of electrochemiluminescence, the reaction is quickly deactivated (fading

image) owing to Au oxide formation, which was prevented using Janus Au-Pt NPs

(107).

3.2.3.2. Probing entities transformation

Owing to the sensitivity of the LSPR of a metallic NP to its free-electron density,

Mulvaney et al. (108) proposed monitoring the shift in LSPR wavelength by

spectroscopic scattering microscopies to image and quantify the flow of (few)

electrons during (dis)charging of Au NPs. The method has since been used to probe

any (electro)chemical reaction that would perturb the electron density of NPs (109,

110) to evaluate the rate of oxidation of ascorbic acid by O2 (111).

The LSPR is also influenced by the refractive index of the chemical environment of

the NPs, which allows imaging the electrocatalytic reactions that modify it, as

illustrated by Long et al. (112) during the oxidation of H2O2 on Au nanorods. The

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nanorod surface, first oxidized to Au hydroxide/oxide, is then reduced back to Au

while oxidizing H2O2, with the two activities represented by different LSPR shifts.

Local refractive index changes following electrocatalytic reactions at nonplasmonic

NPs can also be detected, but they must be deposited on an optically active electrode

that allows such sensitivity, e.g., in plasmonic-based or interference scattering

microscopy. The concept, developed by Tao et al. (28), to obtain local

electrochemical activities of heterogeneous electrodes allowed the establishment of

hydrogen evolution reaction (HER) CV at single Pt NPs (113).

Electrocatalysis of the HER or oxygen evolution reaction (OER), which is critically

important in energy applications, often leads to the formation of gas bubbles. Bubbles

are thought to nucleate and grow in regions supersaturated by gas molecules. Optical

microscopies that can probe bubble production at the micro (114) and nanoscale (115)

enable the identification of the most active catalysts.

Nanobubbles (NBs) were revealed during HER on Au nanoplates by TIR fluorescence

microscopy by tracking the adsorption of a single rhodamine molecule at the

electrolyte-gas interface (Figure 3a). The collected fluorescence intensity additionally

allows NB size estimation (79, 80). Due to their low refractive index, NBs are also

directly detected by label-free microscopy (Figure 3b). Optically undetectable Au-Pt

NPs were revealed from their electrogenerated NBs (116). Superresolution

plasmonic-based (117) or interference-reflection (82) microscopy allows rapid

dynamic mapping of NB nucleation sites on Au or ITO electrodes. The optical

response further provides a dynamic estimation of the size and shape of NBs (or

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contact angle), suggesting that Pt nanocatalysts were rapidly electrically isolated by

NBs (83), and their continuous growth proceeded through spill-over (79).

INSERT FIGURE 3

3.2.4. Superlocalizing physical changes

Controlling operando the deformation or structural alteration of NPs is crucial for the

longevity of electrocatalytic energy conversion devices (85). Such information, often

hidden in electrochemical analysis, except for some stochastic collision experiments

(56), is within the reach of optical imaging via spatial superlocalization in 2D or 3D

of the centroid of the optical pattern of NPs (118) during electrochemical solicitation.

The edge-tracking procedure suggested by Tao and coauthors allows localization of

the contours and thus evaluation of an apparent size of objects with a size comparable

or higher than the diffraction limit (119). The strategy highlighted the reversible

breathing of single Co(OH)2 particles while being electrochemically probed in the

OER region (120).

The motion and orientation of individual electroactive pseudo-2D graphene

microplatelets were optically tracked as they approached and collided with a

microelectrode (Figure 3c). The latter yields variations in the overall electroactive

surface area that correlate with transient variations in the electrochemical current

(121) until they rearrange themselves flat on the electrode (122). The dynamics of the

process are obtained from the rate of angular motion of the platelet.

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3.3 PROBING SUBNANOENTITIES

Optical imaging can image beyond the single NP resolution and probe

transformations at the subunit level. First, an NP is separated into two classical

subunits: the shell, which is in contact with the outer environment, can exhibit a

different reactivity than the NP core. With the help of dedicated optical models, the

contribution of these subunits can be revealed optically. In a second approach,

anisotropic or localized electrochemical processes inside an NP are revealed by a

superlocalization approach (2.2.4).

3.3.1 Surface alteration

Imaging the shift in LSPR wavelength of plasmonic NPs by spectroscopic DFM

allows probing (electro)chemical conversion of the NP shell. The strategy developed

to monitor the deposition of Ag on strongly scattering Au nanostars (123) allows the

detection of the underpotential deposition of Ag on various shaped Au nanocrystals.

An LSPR shift of a few nm corresponds to submonolayer deposition. An optical

voltammogram (Figure 4a) is obtained from the LSPR frequency variations, revealing

the influence of the crystallographic facet orientation on the Ag electrodeposition

potential (124). The electrochemical conversion of the Ag shell to AgCl was

monitored in a similar manner (Figure 2a). The reaction intermediates distinguished

optically from their different plasmonic coupling modes suggest propagation of the

Ag/AgCl interface between the Au core and the chloride electrolyte interface (125).

The reversible electrochemical de/amalgamation of Au NPs by Hg (126, 127) was

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similarly imaged. It first involves saturation of the NP surface by Hg atoms before

their slow diffusion into the core. The reverse process operates in the same way but

with a slower solid diffusion rate for the expelled Hg atoms.

Similarly, Link, Landes et al. (110) distinguished the reversible physical adsorption of

chloride ions on the surface of an Au NP from the irreversible formation of an Au

chloride shell. Then, they generalized the method to probe the electrochemical

adsorption dynamics of various molecules or anions (128).

As discussed in 2.2, the chemical reactivity of the NP surface, not restricted to

plasmonic NPs, was probed using refractive index-based techniques. Plasmonic-based

imaging has been used to differentiate between surface and bulk oxidation (or

reduction) for Au NPs or electrodes (129, 130). Interferometric scattering microscopy

has been more recently introduced to electrochemical studies, although it shows high

imaging sensitivity of various charge transfer processes. Although at the LiCoO2

microparticle but in a real Li-ion battery configuration, it allowed operando dynamic

imaging of local Li ion flow during (des)insertion (Figure 4b), revealing how its

heterogeneity can alter battery operation (131). It could also identify the restructuring

of electrochemical double layers at ITO or Cr nanostructures in iodide electrolyte

(132).

INSERT FIGURE 4

3.3.2 Centroid superlocalization

Edge-tracking procedures (119) were used to evaluate the electrochemical

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deformation of gold nanowires (133) and graphene sheets (44) due to surface stress

and Pauli repulsion, respectively.

The superlocalization of AgCl NPs colliding with a cathodically biased electrode

revealed conversion in multiple motion-reaction steps attributed to loose electrical

connections (Figure 2a). Chloride ions are released locally in multiple steps, each

creating a limited silver metal inclusion within the NP and propelling the NP to a

nearby reactive site (86).

If the displacement of the NP PSF over distances greater than the NP dimension

reveals their physical motion, a slight spatial fluctuation can be attributed to an

asymmetric transformation of the NPs, highlighting the presence of inactive zones

within the NP.

A shift in the centroid of Ag NPs was observed by Willets et al. during their oxidation

(134), suggesting asymmetric dissolution limited by the asymmetric formation of a

nonconductive surface oxide (Figure 2a).

Similarly, the reduction of single PB NPs (98) to Prussian white is not always

complete. The position of the optical centroid depends on the intermediates formed

locally and therefore fluctuates during the conversion (Figure 4c). A microscopy

approach was then proposed to evaluate the propagation of the reaction along the

vertical direction. Since no vertical propagation is detected, conversion is thought to

occur via a shell-to-core model.

Ultimately, Wang and coauthors showed that Fourier transform analysis of the optical

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images enables pushing the superlocalization procedure down to subnanometer

accuracy. By optically imaging the charge separation in Au nanorods subjected to

periodic capacitive charge-discharge cycles, they detected a periodic subnanometer

centroid shift (Figure 4d), suggesting heterogeneous charge accumulation on the Au

surface (96). This unprecedented resolution should unlock the label-free observation

of nanoscale local surface chemistry or the manipulation of single NP local reactive

sites (135).

3.4 SINGLE MOLECULE ELECTROCHEMISTRY

Molecular electrochemistry is a very broad subject involving many new concepts and

techniques, some with an unprecedented level of spatial resolution giving it a new

impetus, such as for the establishment of structure-function relationships at the

single-molecule scale (14, 15, 59, 136–138). In 1995, Fan and Bard first demonstrated

a single-molecule electrochemical measurement. This uses the concept of current

amplification by catalytic redox cycles, which involves repeating the oxidation and

reduction events of a molecule placed between two electrodes (139). To date, high

spatial resolution optical approaches have been developed to capture the intermediate

states of the electrochemical reaction of a single electroactive molecule, such as

surface-enhanced Raman spectroscopy and single-molecule fluorescence

spectroscopy (14, 16, 140, 141). Very recently, these methods have been transposed

to electrochemiluminescence imaging by Feng and coauthors (Figure 5a), showing

how, in complete darkness but by precise control of the chemical reaction between

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electrogenerated reactants, electrodes can turn-on single-photon chemiluminescent

reactions (13). This opens a fascinating area of molecular electrochemistry and

electroanalytical research.

INSERT FIGURE 5

More abundant literature uses the former two optical approaches detailed here. The

tiny optical response during the transition of different redox states of a single

molecule can be followed to probe the electrochemical dynamics. This reveals the

intrinsic mechanism of electron transfer reactions in homogeneous solutions, enabling

the fundamental understanding of the electrochemistry of single molecules.

3.4.1 Surface-enhanced Raman spectroscopy

Single molecule surface-enhanced Raman spectroscopy (SM-SERS) can be used to

directly probe individual heterogeneous electrochemical events in a single molecule

(8, 14, 16, 138, 142). It provides fundamental information about structural changes

and specific behavior of a surface reactive site with respect to a redox couple or to

understand molecular electron transfer mechanisms and intracellular dynamics in

analytical chemistry. Plasmonic nanostructures locally enhancing the Raman intensity

are defined as "hot spots" in SERS in which vibrational information is captured to

determine the redox transient states of target electroactive molecules.

In 2010, SM-SERS was applied for the first time to discover electrochemical events in

a bianalyte system combining two dye molecules, rhodamine-6G (R6G) and Nile blue

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(143). In an open-frame electrochemical cell, the presence of distinct imprinting

modes of a molecule at a hot spot was captured to address redox (on-off) events. In

addition, a thought-provoking question was answered as to whether the "average"

behavior of a bulk system can be recovered from the events of a single molecule.

Identical local conditions can only be extracted from measurements of a single

molecule without averaging electrical or optical properties. In parallel, Van Duyne

implemented SM-SERS to study single electron transfer events (O+ 1e! ⇄ R) of the

dye molecule R6G adsorbed on a silver NP under nonaqueous conditions (144). The

broad local distribution of reduction potentials can be attributed to variations in

molecular orientations and variations in the local surface site or chemical potential of

the R6G-Ag bonding units.

Recently, Wilson and Willets demonstrated the superresolution imaging strategy of

SM-SERS with sub-10 nm accuracy by establishing the spatial relationship between

the centroid of the SERS emission and the corresponding maximum intensity (104,

145, 146). Using this approach, they visualized the specific redox potentials at

different adsorption sites of individual Nile blue molecules on colloidal Ag NPs. The

reversible trajectories of the centroid of the molecules on the surface of the NPs

during a redox cycle were attributed to the location-dependent potentials of the single

electroactive molecule, where the SERS intensity modulates the activation and

deactivation states with oxidation and reduction processes.

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3.4.2 Single molecule fluorescence spectroscopy

The synergistic coupling of electrochemistry with single molecule fluorescence

spectroscopy (SMFS), via confocal laser scanning, TIR, or superresolution

microscopes (15, 59, 137, 147, 148), allows the study of heterogeneous electron

transfer events by simultaneously capturing a quantitative electrochemical signal and

in situ fluorescence images. The key feature of this coupling is to obtain both

temporally and spatially resolved information by following the electron transfer

process. The redox states of the electrofluorochromic compounds at the

single-molecule limit can be determined from the blinking (on/off states) of the

fluorescence response. As the electrode potential varies, the residence time constant in

each of the states (on/off, then ox/red) reflects the rate of the redox transformation and

thus the dynamics of the electrochemical reaction.

In 2006, Bard and Barbara demonstrated for the first time the possibility of studying

single-molecule electron transfer processes by spectroelectrochemistry (149). They

studied hole-injection oxidation events of single molecules of

poly-9,9-dioctylfluorene-cobenzothiadiazole (F8BT), a redox conjugated organic

polymer used in solar cells and flat panel displays, immobilized on an ITO electrode.

As oxidation quenches fluorescence, the electron transfer dynamics are studied as a

function of potential and illumination. If both the excited and ground states of F8BT

can be oxidized, only the ground state oxidation shows a narrow distribution of

fluorescence turn-off potential, revealing its half-wave potential.

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The technique has been extended to the study of more conventional fluorescent probes

in bioimaging. Gooding et al. were the first to report reversible fluorescence switching

of bovine serum albumin (BSA)-conjugated Alexa Fluor 647 redox probes by TIRF

(150). The potential-modulated fluorescence of BSA-Alexa Fluor 647 immobilized on

ITO at the single protein level was studied by measuring the variation in the number

and intensity of fluorescent spots. The observed pH dependence indicates the

involvement of two-electron one-proton transfer in the fluorescence switching

mechanism.

Orrit and coauthors (151) studied the fluorescent readout of redox-sensitive methylene

blue probes at the single-molecule level enhanced by individual gold nanorods

(Figure 5b). MB, a common redox indicator for tissue staining and biosensing,

undergoes a reversible fluorescence change to form colorless methylene blue by

two-electron one-proton transfer. Time traces of the fluorescence flashing of a single

electrogenerated MB molecule are recorded at different potentials. The residence

times in the on/off states are evaluated by a step detection algorithm. The distribution

of these residence times at each potential is used to evaluate the half-wave potential of

single molecule electrochemical switching from the Nernst equation.

4. PERSPECTIVES AND CONCLUSION

This review has shown how advanced optical microscopies are now able to image a

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wide range of electrochemical phenomena with unprecedented temporal and spatial

resolutions (below the diffraction limit), up to the single object level with subentity,

subnanoparticle or single molecule sensitivity. By providing quantitative descriptors

complementary to electrochemical signals, they have unraveled old problems while

revealing new ones in the different fields explored by electrochemistry (sensors,

electroanalysis, corrosion, electrocrystalization, energy conversion and storage,

electrocatalysis, etc.).

First developed through model systems, they are now shifting to materials and

configurations focused on real-world applications, where they can be exploited to

precisely locate heterogeneous electrochemical processes, distinguish domains

(electrode regions, nanoobjects, etc.) of different structure/composition and therefore

distinguish competing chemical routes, or identify the origin of problems to fix. As

definitely the most intuitive platform to see operando, optical microscopies should

become a routine electroanalytical tool to evaluate the performance of electroactive

materials and rationalize their design or degradation. An even deeper degree of

understanding can be reached from their simple implementation with complementary

structural and chemical analyses such as spectroscopy (UV-vis or Raman, as well as

the promising surface-enhanced IR) or within multicorrelative microscopies

combining, e.g., local electrochemical probes and in situ TEM. Particularly,

approaches combining optical visualization within complementary electrochemical

local probing, e.g. by SECCM (57, 60), will enable the generation of large sets of

correlated optical and electrochemical data. It should become a powerful approach for

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benchmarking wide range of electrochemical situations.

The generalization of these explorations to broader electrochemical situations also

implies seeing with greater sensitivity (e.g., iSCAT (10, 132) or photothermal

microscopes) more rapidly in more complex media (seeing through fog is within

reach) or in real-world systems (optical fiber explorations). It is also necessary to

generalize the nature of current collectors (optoelectrodes) providing sensitive optical

detection ensuring homogeneous (electro)chemical contact with the objective of

studying minimal electrocatalytic activity, for which transparent carbon- or

graphene-based electrodes are promising. Finally, the thousands of data per image,

even tenfold with complementary spectroscopic data, promise to unlock many

structure-function understandings. The use of artificial intelligence will be crucial to

achieve faster automated postprocessing, e.g., object identification by deep learning

(152), or recognition of electrochemical behavior and for the removal of unnecessary

information to optimize data storage and processing in real time.

ACKNOWLEDGMENTS

F.K. acknowledges support from the European Union’s Horizon 2020 Research and

Innovation Programme under Marie Skłodowska-Curie MSCA-ITN Single-Entity

Nanoelectrochemistry, SENTINEL [812398]. J.-F.L. and F.K. acknowledge the

Université de Paris and CNRS for financial support. W. Wang and H. Wang

acknowledge the National Natural Science Foundation of China (Grants 21925403,

21904062 and 21874070) and the Excellent Research Program of Nanjing University

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(Grant ZYJH004) for financial support.

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FIGURE CAPTIONS

Figure 1. (a) Schematic illustration of the plasmonic-based electrochemical

impedance imaging technique of action potentials in single neurons. A micropipette is

patched on single neurons cultured on the surface to trigger action potentials, which

are recorded by patch clamp electronics and plasmonic imaging. Adapted with

permission from Reference (22). Copyright 2017, John Wiley & Sons. (b) Schematic

illustration of a typical electrochemiluminescence imaging technique for visualizing

the latent fingerprints on electrode surfaces with negative and positive modes.

Adapted with permission from Reference (29). Copyright 2012, John Wiley & Sons.

(c) Superlocalization of Zn dendrite nucleation and growth monitored by dark-field

microscopy in a Zn aqueous battery configuration. Adapted with permission from

Reference (54). Copyright 2021, Elsevier.

Figure 2. (a) Summary of optical microscopy studies reporting single silver-based NP

electrochemistry grouped into three main categories: growth, dissolution and

conversion, with corresponding references. Adapted with permission from Reference

(3). Copyright 2021, John Wiley & Sons. (b) Quantitative light scattering monitoring

of silver NP deposition and stripping voltammetry. The optical intensity transients

(extracted in ROIs) are quantitatively converted into single NP currents and

optovoltammograms. Adapted with permission from Reference (64). Copyright 2018,

John Wiley & Sons. (c) Optical transmittance monitoring of WO3 NP electrochemical

conversion. The different optical transients reveal heterogeneous Li-ion insertion in

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single NPs and aggregates. Adapted with permission from Reference (94). Copyright

2019, American Chemical Society.

Figure 3. (a) Imaging of gas nanobubbles nucleating and growing upon

electrocatalytic water splitting in the vicinity of four nanocatalysts by TIR

fluorescence microscopy. Adapted with permission from Reference (79). Copyright

2018, National Academy of Sciences. (b) H2 nanobubbles equivalently detected at

single Pt nanocatalysts by interference reflection microscopy. The optical data are

further exploited to estimate the evolution of the contact angle of single NBs during

the growth process. Adapted with permission from Reference (83). Copyright 2021,

American Chemical Society. (c) Optical tracking of the graphene platelet impact

event and further dynamic rotation at a polarized microinterface. Adapted with

permission from Reference (122). Copyright 2021, American Chemical Society.

Figure 4. Operando optical screening enables subentity studies. (a) Optically inferred

voltammetry tracking facet-dependent underpotential deposition of silver atoms on

single gold truncated octahedral nanocrystals. Adapted with permission from

Reference (124). Copyright 2020, CC BY 4.0. (b) Optical image showing the front of

the phase transition in the LixCoO2 cathode particle, from which the charge-discharge

dynamics are revealed operando in a real Li-ion battery. Adapted with permission

from Reference (131). Copyright 2021, Springer Nature. (c) The optical centroid

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motion of a single Prussian blue NP during oxidation/reduction cycles reveals local

transformations or inactive sites. Because the centroid is moving back to its initial

position, the conversion is reversible. Adapted with permission from Reference (98).

Copyright 2021, CC BY-NC 3.0. (d) Ultimate tracking resolution: the electrochemical

charging of single Au nanorods results in subnanometer optical centroid motion

owing to local electron accumulation. Reproduced with permission from Reference

(153). Copyright 2019, American Chemical Society.

Figure 5. Electrochemistry with a single-molecule fluorescence readout. (a) Principle

of single electrochemiluminescent event observation enabling single molecule

luminescence imaging, without illumination, of arrays of nanoband electrodes. The

dilution of both the dye and coreactant electrogenerated intermediate imposes a single

reaction event, i.e. the formation of the excited dye molecule further emitting a single

photon, located where the reaction was initiated during the image snapshot. Adapted

with permission from Reference (13). Copyright 2021, Springer Nature. (b)

Schematic showing the local fluorescence emission under plasmon-enhanced

photoactivation of the electrogenerated fluoroactive form of a single immobilized

electrofluorophore. The red/ox proportion of the single molecule is obtained from

blinking (right part) of the fluorescence signal at a fixed electrode potential. Adapted

with permission from Reference (151). Copyright 2021, John Wiley & Sons.

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FIGURE 1

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FIGURE 2

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FIGURE 3

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FIGURE 4

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FIGURE 5